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Related Experiment Videos

A new method for protein coexpression in Escherichia coli using two incompatible plasmids.

W Yang1, L Zhang, Z Lu

  • 1College of Life Sciences, Peking University, Beijing 100871, People's Republic of China.

Protein Expression and Purification
|August 3, 2001
PubMed
Summary

Two incompatible plasmids can coexist in Escherichia coli under dual antibiotic selection, enabling efficient coexpression of foreign proteins like human DNA fragmentation factor subunits.

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Area of Science:

  • Molecular Biology
  • Biotechnology
  • Microbial Genetics

Background:

  • Incompatible plasmids typically cannot stably coexist in a single bacterial cell.
  • Previous methods faced limitations in coexpressing multiple proteins within Escherichia coli.

Purpose of the Study:

  • To investigate the stable coexistence of incompatible plasmids in Escherichia coli under selective pressure.
  • To develop a novel method for coexpressing foreign proteins using two incompatible plasmids.

Main Methods:

  • Co-transforming E. coli BL21(DE3) with two incompatible plasmids (pET-21a and pET-28a) conferring ampicillin and kanamycin resistance, respectively.
  • Applying simultaneous selection pressure with both antibiotics.
  • Inducing coexpression of human DNA fragmentation factor (DFF) subunits (DFF45 and DFF40) using isopropyl β-D-thiogalactoside.

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Main Results:

  • Achieved stable coexistence of incompatible plasmids in E. coli for over 14 hours under dual antibiotic selection.
  • Successfully coexpressed DFF45 and DFF40 subunits, yielding soluble protein fractions for both.
  • Demonstrated that coexpression resulted in soluble DFF40, unlike its insoluble form when expressed alone.
  • The coexpressed DFF exhibited functional caspase-activated DNase activity.

Conclusions:

  • Stable coexistence of incompatible plasmids is achievable in E. coli under specific selective conditions.
  • This method enables efficient coexpression of foreign proteins, overcoming challenges with single-subunit insolubility.
  • The coexpression system provides a viable platform for producing functional multi-subunit protein complexes.